Introduction

Current media formulations used to culture undifferentiated pluripotent human ES and induced pluripotent stem (iPS) cells require media replenishment every day and at least 1 media exchange on the weekend. The costs in media reagents and human resources become prohibitively expensive as increasing numbers of core labs and consortiums are focused on generating diseased human iPS cell models. PluriSTEM™ Human ES/iPS Medium is a small molecule based medium that enables weekend-free culture of human pluripotent stem cells and allows for media exchanges every other day without compromising the morphology or long term functionality of pluripotent stem cells. Pluripotent cells maintained in this moderate feeding regiment exhibited high cell health with minimal spontaneous differentiation, expressed high levels of pluripotency markers (NANOG, OCT3/4, SOX-2, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), retained differentiation potential and possessed a normal karyotype.

Methods

Preparation of Coated Plates

Expansion of pluripotent human ES and iPS cells with PluriSTEM medium requires cultureware that are coated with Matrigel. Below are general guidelines for the coating of 6-well plates and culture flasks with Matrigel:

Table 1: Volumes recommended for coating cultureware:

CultureWare

Coating Volume (mL)

Surface Area (cm2)

12 well plate

0.5 mL/well

2.0

6 well plate

1.5 mL/well

9.6

T25 flask

3 mL

25

T75 flask

8 mL

75

Thaw Matrigel on ice. Keep on ice and use pre-cooled medium and pipettes to avoid gelling of the ECM gel.

IMPORTANT: Do not thaw Matrigel at temperatures higher than 15°C to avoid gelling.

Dilute the Matrigel 1:20 with cold DMEM/F12 medium. For example, to every 0.5 mL Matrigel, add 9.5 mL cold DMEM/F12 medium for a total volume of 10 mL. Scale according to the volumes required.

Cover the cultureware with the recommended volumes (see Table 1). Swirl the culture plates to spread the Matrigel evenly across the surface of the plate. Incubate at room temperature for at least 1 hour or 2 – 8°C overnight. If not used immediately, store coated cultureware at 2-8°C until ready to use.

Note: If not used immediately, Matrigel coated culturewares should be sealed with parafilm to prevent evaporation and can be stored at 2 – 8°C for up to one week.

Prior to seeding the cells, bring the plate back to room temperature, remove the coating solution and add an appropriate volume of PluriSTEM Human ES/iPS Medium. IMPORTANT: Do not allow the flask to dry out.

Preparation of Dispase II

Pluripotent human ES and iPS cells maintained in PluriSTEM may be enzymatically passaged using Dispase II. MilliporeSigma provides Dispase II as a ready to use 1 mg/mL stock solution. We recommend that Dispase II (1 mg/mL) be aliquoted into smaller working volumes and store at -20°C for up to 4 months from date of receipt. Frozen aliquots may be thawed and stored at 2 – 8°C for up to 2 weeks. Avoid multiple freeze thaw cycles to maintain proper enzymatic activity.

Transition of Human ES/iPSCs to PluriSTEM Media

(Applicable for both Feeder-Based and Feeder-Free Media Systems)

One to two days prior to passaging, exchange the media with 3 mL PluriSTEM per well of the 6 -well plates containing human pluripotent ES or iPS cells. Exchange with fresh PluriSTEM the next day. There should be minimal cell loss observed with the transition to PluriSTEM medium.

Coat the 6-well plates with 1:20 dilution of Matrigel (1.5 mL per well) (see “Preparation of Coated Plates”). Swirl the culture plates to spread the Matrigel evenly across the surface of the plate. Incubate at 2 – 8°C overnight or at room temperature for 1 – 2 hours before use.

On the day of passaging, acclimate Matrigel coated plates for 1 hour at room temperature. After 1 hour, remove the Matrigel coating. Add 2 mL PluriSTEM media to each well. Set plate aside until cells are ready to be passaged.

Use a dissection microscope to visually inspect the plate containing human pluripotent cells to be passaged. Inspect the colonies for areas of spontaneous differentiation.

Note: Areas of spontaneous differentiation are characterized as phase-bright, highly dense areas with irregular borders, non-uniform cell morphologies and cell types and are typically localized either in the center of the colonies or along the edges between colonies.

Use a sterile p200 pipette tip attached to a p200 pipetman to scrape away areas of spontaneous differentiation. Be discriminating and scrape away any areas that harbor a hint of differentiation.

Note: It is critical to start with high quality undifferentiated human ES and iPS culture. When maintained correctly, spontaneous differentiation should be <1 – 5% in PluriSTEM culture. However, in the event that the starting culture contains large areas of differentiation, the colonies may still be rescued using PluriSTEM. Be discriminating and scrape away any areas that harbor a hint of differentiation even if it means sacrificing the majority of the colonies. Remaining undifferentiated colonies will recover and proliferate in PluriSTEM to repopulate into high quality pluripotent colonies.

Aspirate the medium containing the scrapped areas from the well. Rinse with 2mL per well of DMEM/F-12 medium or 1X PBS.

Add 1 mL Dispase II (1 mg/mL) per well of the 6-well plate containing pluripotent human ES or iPS cells to be passaged.

Incubate at 37°C for 6 – 7 minutes. After incubation, visually inspect the colonies under a microscope. The edges of the colonies may appear slightly rounded up and folded back but the overall colony should still be attached to the plate.

Aspirate the Dispase II and gently rinse each well two times with 2 mL 1X PBS or DMEM/F12 medium to remove any residual Dispase II solution. Aspirate after each rinse.

Use a 5 mL serological pipette to collect the cell aggregates to a 15 mL conical tube. Minimize pipetting up and down as this may break up the colonies to suboptimal small pieces. The process of transferring the cell aggregates to the 15 mL conical tube should be sufficient to break the colonies to sufficient size.

Rinse the wells with an additional 2 mL of PluriSTEM medium per well to collect any remaining cell aggregates. Add the rinse to the 15 mL conical tube.

Aspirate the supernatant. Resuspend the cell aggregates in an appropriate volume of PluriSTEM for passaging. Do not pipette the cell aggregates more than 1 – 2 times with a 5 mL serological pipette, taking care not to break the aggregates into single cell suspensions. For example, for a 1:5 split ratio, resuspend the cell aggregates in 5 mL total PluriSTEM. For a 1:3 split ratio, resuspend the cell aggregates in 3 mL total PluriSTEM.

Note: For a confluent culture, a split ratio of 1:5 – 1:6 is recommended. For less confluent cultures, a 1:3 – 1:4 split ratio may be more optimal. However as culture techniques and cell lines may vary, it is recommended that the users set up a titration of split ratio ranging from 1:3 to 1:6 to determine the optimal split density.

Aliquot 1 mL of the appropriately diluted cell aggregates into the Matrigel coated plates containing 2 mL PluriSTEM medium that had been set aside from step 2. Total volume per well = 3 mL.

Place the plate in a 37°C incubator. Agitate the plate gently from side to side and forward and backwards to ensure that the cell aggregates are evenly distributed across the surface of the well. Incubate in a 37°C incubator.

After 10 – 15 minutes, visually inspect the plate to ensure that newly passaged cell aggregates are evenly distributed across the surface of the well. Plates that have not been properly agitated may have cell clumps aggregating toward the center of the wells. This uneven distribution at the center may later cause spontaneous differentiation of human ES/iPS cells. In the event clumps are not evenly distributed, agitate the plate gently from side to side and forward and backwards for a longer extended time.6

The next day, replace with 3 mL per well of fresh PluriSTEM medium.

Monitor and exchange with 3 mL fresh PluriSTEM media daily. Depending upon the split ratio used, cells are typically ready for enzymatic passaging in 4 – 6 days. For example, for 1:5 – 1:6 split ratio, cells may be ready to be passaged within 5 – 6 days whereas for a more conservative split ratio of 1:3, cells may require passaging within 3 – 4 days.

Cultures should be fed with 4 mL PluriSTEM on Friday to allow sufficient media to sustain the cells over the weekend. Medium exchanges during the weekend are not necessary.

When pluripotent cultures have been maintained in PluriSTEM for at least 3 passages, media exchanges may be transitioned to every other day. Monitor cell health daily to ensure that every other day exchange does not affect the cell health and quality of the colonies.

Single Cell Passaging of Human ES/iPSCs using Accumax

Coat new 6-well plates with 1:20 dilution of Matrigel (1.5 mL per well) (see “Preparation of Coated Plates”). Swirl the culture plates to spread the Matrigel evenly across the surface of the plate. Incubate 2 – 8°C overnight or at room temperature for 1 – 2 hours before use.

On the day of passaging, acclimate Matrigel coated plates for 1 hour at room temperature. After 1 hour, remove the Matrigel coating. Add 2 mL PluriSTEM media to each well. Set plate aside until cells are ready to be passaged.

One hour before the cells are to be passaged, add ROCK Inhibitor, Y-27632 to each well of the 6-well plate at a final concentration of 10 μM.

After 1 hour, use a dissection microscope to visually inspect the plate containing human pluripotent cells to be passaged. Inspect the colonies for areas of spontaneous differentiation.

Use a sterile p200 pipette tip attached to a p200 pipetman to scrape off areas of spontaneous differentiation. Be discriminating and scrape away any areas that harbor a hint of differentiation.

Note: It is critical to start with high quality undifferentiated pluripotent human ES and iPS culture. Pluripotent colonies cultured in PluriSTEM typically have <1% spontaneous differentiation when maintained correctly. However, in the event that the starting culture contains large areas of differentiation, the colonies may still be rescued using PluriSTEM. Be discriminating and scrape away any areas that harbor a hint of differentiation even if it means sacrificing the majority of the areas within a colony. So long as the remaining small colony pieces are of high quality and undifferentiated, PluriSTEM will still rescue these low density cultures. Remaining undifferentiated pieces will recover and proliferate in PluriSTEM to repopulate into high quality undifferentiated pluripotent colonies.

Aspirate the medium containing the scrapped areas from the well. Rinse with 2mL per well with DMEM/F-12 medium or 1X PBS.

Aspirate and replace with 1 mL of Accumax per well of a 6-well-plate. Incubate at 37°C for 8-10 minutes.

Note: Different cell lines may require different incubation time. It is thus important to monitor the cell dissociation. Accumax treatment should be stopped when the cells start to dissociate and holes start to appear within colonies.

Quench the Accumax reaction by adding 1 mL PluriSTEM for each mL of Accumax used. Gently detach cells using a sterile 1000-μL pipette tip. Cells should be easily dislodged.

Collect the dissociated cells to a 15 mL conical tube. Rinse the wells with an additional 2 mL of PluriSTEM medium to collect any remaining cells. Add the rinse to the 15 mL conical tube.

Count the number of cells using a Scepter or hemacytometer. Ensure that the cells are in a single cell suspension. Determine the cell viability using Trypan Blue exclusion.

Set up a titration of different cell densities ranging from 0.5 – 1 x 104 cells/cm2. This corresponds to 50,000 – 100,000 cells per well of a Matrigel-coated 6-well plate in PluriSTEM medium containing 10 μM ROCK Inhibitor, Y-27632.

The next day, replace with fresh PluriSTEM media. The ROCK Inhibitor, Y-27632 is no longer required from this step forward. Replace with fresh PluriSTEM medium daily (3 mL volume per well).

After 6 – 7 days, human ES/iPS cells should be ready for passaging. Human ES/iPS cells are ready for passaging when the colonies are large with centers that are dense and phase-bright compared to their edges. Colonies may also start to merge, covering 80-90% of the surface of the wells.

Results

Figure 1. Every Other Day and Weekend Free Media Exchanges with PluriSTEM. H9 cells were maintained in PluriSTEM for 12 passages during which time media was exchanged every other day and not performed on the weekend. Cells retained characteristic pluripotent morphology (i.e. homogeneous round colonies with defined borders). Guava flow analyses indicate high expression levels of pluripotent markers OCT-4 and SSEA-4 and an absence of staining for SSEA-1.

Figure 4. Robust single cell passaging with PluriSTEM. H9 hESCs cultured in PluriSTEM for 22 passages were dissociated into single cells using Accumax. One hour before dissociation, 10 μM ROCK Inhibitor was added. 100,000 singly dissociated cells were seeded into each well of a matrigel-coated 6-well-plate. After 6 days in PluriSTEM, singe cells had formed distinct pluripotent colonies (A). Single cells continued to form distinct pluripotent colonies after undergoing four rounds of single cell passaging using Accumax (B).